During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Angiogenesis is the development of new blood vessels from pre-existing ones. It is a complex multifaceted process that is essential for the adequate functioning of human organisms. The investigation of angiogenesis is conducted using various methods. One of the most popular and most serviceable of these methods in vitro is the short-term culture of endothelial cells on Matrigel. However, a significant disadvantage of this method is the manual analysis of a large number of microphotographs. In this regard, it is necessary to develop a technique for automating the annotation of images of capillary-like structures. Despite the increasing use of deep learning in biomedical image analysis, as far as we know, there still has not been a study on the application of this method to angiogenesis images. To the best of our knowledge, this article demonstrates the first tool based on a convolutional Unet++ encoder–decoder architecture for the semantic segmentation of in vitro angiogenesis simulation images followed by the resulting mask postprocessing for data analysis by experts. The first annotated dataset in this field, AngioCells, is also being made publicly available. To create this dataset, participants were recruited into a markup group, an annotation protocol was developed, and an interparticipant agreement study was carried out.

Microvesicles (MVs) are plasma extracellular vesicles ranging from 100 (150) to 1000 nm in diameter. These are generally produced by different cells through their vital activity and are a source of various protein and non-protein molecules. It is assumed that MVs can mediate intercellular communication and modulate cell functions. The interaction between natural killer cells (NK cells) and endothelial cells underlies multiple pathological conditions. The ability of MVs derived from NK cells to influence the functional state of endothelial cells in inflammatory conditions has yet to be studied well. In this regard, we aimed to study the effects of MVs derived from NK cells of the NK-92 cell line stimulated with IL-1β on the phenotype, caspase activity, proliferation and migration of endothelial cells of the EA.hy926 cell line. Endothelial cells were cultured with MVs derived from cells of the NK-92 cell line after their stimulation with IL-1β. Using flow cytometry, we evaluated changes in the expression of endothelial cell surface molecules and endothelial cell death. We evaluated the effect of MVs derived from stimulated NK cells on the proliferative and migratory activity of endothelial cells, as well as the activation of caspase-3 and caspase-9 therein. It was established that the incubation of endothelial cells with MVs derived from cells of the NK-92 cell line stimulated with IL-1β and with MVs derived from unstimulated NK cells, leads to the decrease in the proliferative activity of endothelial cells, appearance of the pan leukocyte marker CD45 on them, caspase-3 activation and partial endothelial cell death, and reduced CD105 expression. However, compared with MVs derived from unstimulated NK cells, a more pronounced effect of MVs derived from cells of the NK-92 cell line stimulated with IL-1β was found in relation to the decrease in the endothelial cell migratory activity and the intensity of the CD54 molecule expression on them. The functional activity of MVs is therefore mediated by the conditions they are produced under, as well as their internal contents.

Natural killer cells (NK cells) represent a group of lymphocytes of innate immunity. In addition to NK cells of peripheral blood, tissue-resident populations are described. NK cells of the decidual envelope (decidual NK cells) represent one of the local NK cell populations. Decidual NK cells differ in phenotype and function from peripheral blood NK cells. These cells have, mainly, regulatory functions. At the same time they retain the ability to perform cytotoxic effects. In the uterus, NK cells are located closely to the cells of fetal origin, i.e., trophoblast cells, which differentiate from the outer layer of the invading blastocyst. The purpose of the review article was to analyze the literature data on the studies of the molecular interactions between NK cells and trophoblast cells, as well as potential means of regulating these interactions. The review presents currently available data on receptor-mediated effects (due to adhesion molecules and cytotoxic receptors) and distant interactions (involving cytokines, chemokines and growth factors secreted by the both cell types) between NK population and trophoblast cells. The receptors regulating contacts of NK cells and trophoblast cells with extracellular matrix are also considered. The review provides information on activation of signaling pathways in NK cells and trophoblast cells resulting from their interaction with each other and components of the extracellular matrix. Currently, the molecular mechanisms regulating the NK cell functions and their interaction with trophoblast cells have not been studied sufficiently. The authors attempted to consider molecular regulation of the functional activity of NK cells mediated by the molecular complex of RNA polymerase II. We also describe participation of cyclin-dependent CDK8/19 kinases which comprise a part of the mediator complex which provides functioning of immune cells. The data on the participation of CDK8/19 in regulation of intracellular signaling pathways, as well as influence of CDK8/19 on the NK cell functions, are considered. Summarizing the data presented in the literature, one may emphasize that there is an extensive mutual influence of NK cells and trophoblast cells in decidual lining of uterus during pregnancy, thus leading to a changes in phenotype and functions of these cells. Experimental studies are required on the contribution of molecular mechanisms involved in transcription and translation processes to the biology of NK cells, and their role in maintaining interactions between NK cells and trophoblast cells, including the pathways involving CDK8/19.

Natural killer cells (NK cells) exert cytotoxicity towards target cells in several ways, including the expression of apoptosis-mediating ligands (TRAIL, FasL). In addition, NK cells themselves may be susceptible to apoptosis due to the expression of TRAIL receptors. These receptors include TRAIL-R1 (DR4), TRAIL-R2 (DR5), capable of inducing apoptosis, and TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), the so-called “decoy receptors”, which lack an intracellular domain initiating activation of caspases. Of particular interest is the interaction of uterine NK cells with cells of fetal origin, trophoblasts, which are potential targets for natural killer cells to carry out cytotoxicity. The aim of this work was to evaluate the expression of proapoptotic receptors and their ligands as well as CD107a expression by NK cells in a model of interaction with trophoblast cells. To evaluate NK cells, we used cells of the NK-92 line; cells of the JEG-3 line were used as target cells. The cytokines IL-1β, IL-15, IL-18, TNFα, IL-10, TGFβ and conditioned media (CM) of the first and third trimester chorionic villi explants were used as inducers. We established that cytokines changed the expression of apoptotic receptors by NK cells: in the presence of TNFα, the amount and intensity of Fas expression increased, while in the presence of TGFβ, the amount and intensity of expression of the DR5 receptor decreased. Soluble chorionic villi factors alter the expression of TRAIL and FasL by NK-92 cells, which can reflect the suppression of the TRAIL-dependent mechanism of apoptosis in the first trimester and stimulating the Fas-dependent mechanism in the third trimester. In the presence of trophoblast cells, the expression of TRAIL and DcR1 by NK cells was reduced compared to intact cells, indicating an inhibitory effect of trophoblast cells on NK cell cytotoxicity. In the presence of chorionic villi CM and trophoblast cells, a reduced number of NK-92 cells expressing DR4 and DR5 was found. Therefore, soluble factors secreted by chorionic villi cells regulate the resistance of NK cells to death by binding TRAIL, likely maintaining their activity at a certain level in case of contact with trophoblast cells.